This proposal is a resubmission of our competing renewal application of our current project on the role of Na+-K+-Cl- cotransporter isoform1 (NKCC1) in cerebral ischemic damage. The long-term goal of the research is to understand the role of ion transport proteins in disruption of ion homeostasis following ischemia and to determine whether these ion transport proteins are potential targets for developing more effective stroke treatments. NKCC1 transports Na+, K+, and Cl- ions into cells under physiological conditions, and is important in regulation of intracellular Na+ and Cl-, cell volume, and K+ uptake in the central nervous system. In the past funding period, we found that overstimulation of NKCC1 activity following ischemia plays a role in ischemic cell death. For example, NKCC1 activity is up-regulated via protein phosphorylation following oxygen and glucose deprivation (OGD) and transient focal ischemia. Pharmacological inhibition or genetic ablation of NKCC1 is neuroprotective in both in vitro and in vivo ischemia. However, the cellular mechanisms underlying the role of this protein in ischemic damage are not fully understood. In the current funding period, we further discovered that NKCC1 activity plays a role not only in intracellular Na+ and Cl- overload, it also contributes to a delayed, secondary Ca2+ rise in astrocytes and neurons during reoxygenation (REOX) following OGD. Our preliminary study suggests that this pathological Ca2+ entry was mediated by reversal of Na+/Ca2+ exchange (NCXrev). The NKCC1-mediated intracellular Na+ overload in part triggered the NCX in the reverse mode of operation. We found an increased Ca2+ uptake by endothelium reticulum (ER) and mitochondria during REOX. Intriguingly, inhibition of NKCC1 activity attenuated Ca2+ accumulation in ER and mitochondria, significantly reduced loss of (m, and Cyt. C release. These data demonstrate that the ion transport proteins (NKCC1 and NCXrev) are important in ischemic cell damage. In the next stage of the project, we will further examine the molecular mechanisms underlying the role of NKCC1 and NCXrev in neuronal and astroglial damage using in vitro and in vivo models of ischemia. PUBLIC HEALTH RELEVANCE: This proposal is to study the role of Na+-K+-Cl- cotransporter isoform1 (NKCC1) in cerebral ischemic damage. The long-term goal of the research is to understand the role of ion transport proteins in disruption of ion homeostasis following ischemia and to determine whether these ion transport proteins are potential targets for developing more effective stroke treatments.